Use of 5-Enolpyruvylshikmate-3-Phosphate Synthase Encoding Gene for Typing of Staphylococcus aureus Isolated from Skin and Urinary Tract Infections of Human

Objective(s): Staphylococcus aureus is both a successful human commensal and a major pathogen. In this study we investigated the genetic diversity of 26 S. aureus isolates recovered from human skin and urinary tract infections. Materials and Methods:Typing procedure for the studied S. aureus isolates was performed based on PCR amplification of the aroA gene, which encodes the enzyme 5-enolpyruvylshikmate-3-phosphate synthase (EPSPS) that involves in aromatic amino acid biosynthesis, and restriction fragment length polymorphism (RFLP) analysis of the product. Results:All S. aureus isolates produced a single PCR amplification product of 1,153 bp. Digestion of the PCR products with the TaqI endonuclease revealed four different aroA gene patterns designated as A, B, N and H according to the nomenclature system of previous studies. In general, 80.77% of the studied isolates displayed type N, 7.69% were type B, 7.69% were type H and 3.85% displayed type A. Conclusion:Divergent aroA types were detected among S. aureus isolates from skin and urinary tract infections. The results showed that urinary tract infections were contaminated by S. aureus isolates with identical banding patterns (A), while isolates recovered from skin infections had different aroA types. This study also indicates that aroA genotypes vary not only from region to region, but also in individual hosts within a region.


Introduction
Staphylococcus aureus is an increasing public health concern because of the rapid evolution and spread of virulent/resistant clones in humans and animals. This organism is the main etiological agent and the most frequently isolated microorganism in various skin and soft tissue infections (SSTIs) reported from different parts of the world (1)(2)(3)(4)(5)(6). Urinary tract infections (UTIs) are also among the most common infectious diseases diagnosed in outpatients as well as in hospitalized patients and S. aureus is one of the causative agents (7). The molecular mechanisms by which S. aureus causes skin and urinary tract infections remain poorly understood. Human skin gives physical protection partly because it is composed of tightly associated epithelial cells. These cells are covered by a highly cross-linked layer of keratin which is normally impenetrable to bacteria. Additionally, skin displays microbicidal activity via an array of bioactive molecules, among which antimicrobial peptides (AMPs) are of critical importance to host defense (8,9). Skin fatty acids are also potent bactericidal agents and help to prevent colonization of potential pathogens. Resistance to human skin innate defenses is crucial for survival and carriage of S. aureus, a common cutaneous pathogen and nasal colonizer. It must be noted that specific tissue environment is decisive for the differentiation of Staphylococci (10). In a study, van Leeuwen et al (2005) showed that the presence (combinations) of virulence factors plays an important role in host or even tissue specificity in S. aureus infections (11). In a hospital setting, knowledge of the risk factors for SSTIs, clonal spread and resistance pattern are particularly useful in patient management and maintenance of infection prevention measure (12).
There are few data concerning the genotypic characteristic of S. aureus obtained from skin and urinary tract infections. Population analyses based on molecular characterization have proven to be useful for establishing the relationship between clinical isolates responsible for defined diseases. On the other hand, molecular strain typing of microorganisms is now recognized as an essential component of infection control program and to trace the source of infection and routes of transmission. Many molecular methods have been used in epidemiological investigations into human staphylococcal infections such as pulsed-field gel electrophoresis (PFGE) (13,14), restriction fragment length polymorphism analysis (RFLP) (15,16), random amplification of polymorphic DNA (RAPD) (17), multilocus sequence typing (MLST) (18) and spa typing (19). Indeed, the sensitivity and performance of polymerase chain reaction (PCR) based typing systems make them useful tools for the study of S. aureus of animal and human origin, and for the monitoring of the bacterium's diffusion. The aroA gene amplification has been considered a simple and rapid method for typing S. aureus (20). aroA gene encodes the enzyme 5-enolpyruvylshikmate-3phosphate synthase (EPSPS), a key enzyme in the shikimate pathway, which catalyzes phosphoenolpyruvate (PEP) and shikimate-3-phosphate (S3P) to form 5enolpyruvylshikmate-3-phosphate (EPSP) and inorganic phosphate. Two classes of EPSPS have been identified that share less than 50% amino acid identity (21). Class I EPSPS are naturally sensitive to glyphosate, generally identified from plants and bacteria. In contrast to class I, class II EPSPS usually has a natural tolerance of glyphosate and a high affinity for PEP. Class II EPSPS was identified from some bacteria (e.g., Pseudomonas sp. strain PG2982 (21), and Agrobacterium tumefaciens sp. strain CP4). In this study, we have utilized the aroA gene typing in discriminating unrelated S. aureus strains recovered from human urine and skin infections.

Materials and Methods
Origin of the bacterial isolates Twenty-six S. aureus isolates were obtained from urinary tract (n= 16) and skin wound infections (n= 10) in Urmia region, West Azerbaijan province, Iran. The S. aureus strains were isolated from the site of skin infection and from the mid urine samples. The skin swabs were taken from the site of skin infection and immediately streaked on mannitol salt agar plates. After incubation for 24 hr at 37 ○ C, colonies suspected as S. aureus were isolated and purified on sheep blood agar plates. The biochemical tests were used to confirm the species identification of the S. aureus isolates. The diagnosis of a symptomatic UTI in this study required the presence of fever >38 ○ C and dysuria. Cytological examination of the urine required >10 4 leucocytes/ml. Bacterial colonies from the urine were counted and the microbial flora was identified. Recovery of >10 5 bacteria/ml from a voided specimen or >10 4 bacteria/ml from a catheter specimen was required. Diagnosis of S. aureus UTI required the presence of the organism in pure or predominant (>95%) culture.

DNA isolation for PCR assay
Chromosomal DNA was extracted by incubating cells grown overnight in an agar plate with Genomic DNA purification kit (Fermentas, Germany). S. aureus ATCC 29213 was included as a positive control in both PCR and PCR-RFLP assays. For the negative control, sterile water was added instead of nucleic acids.

Detection of the aroA gene
The primers used for analysis of the aroA gene of S. aureus by PCR-RFLP were constructed according to the method described by Marcos et al (1999) (20). The sequence of forward primer used for amplification was 5′-AAG GGC GAA ATA GAA GTG CCG GGC-3′ and the reverse primer was 5′-CAC AAG CAA CTG CAA GCA T-3′. The PCR reaction was carried out in a final volume of 50 μl reaction containing 25 μl of 2X master mix (CinnaGen, Iran), 0.8 μM of each primer, and 5 μl of template DNA. For the negative control, sterile water was added instead of nucleic acids. DNA amplification was carried out with the following thermal profile: initial denaturation at 94 °C for 2 min; 32 cycles, each consisting of a 1-min denaturation step at 92 °C, 1-min annealing step at 63 °C, and 1.5min extension step at 72 °C (22). The reaction was completed by incubating at 72 °C for 10 min. Amplified products were separated by agarose gel electrophoresis (1.2% agarose containing 0.5 mg ethidium bromide in 0.5X TBE electrophoresis buffer) at 80 V for 1 hr and photographed under UV illuminator.

Molecular typing by aroA PCR-RFLP
Enzymatic digestion of the aroA amplified product was performed with 5U of TaqI (Fermentas, Germany) as previously described (22). The resulting fragments were electrophoresed on a 1.2% agarose gel at 100 V for 1 hr, stained with ethidium bromide (0.5 µg/ml), and visualized under UV light. Based on the nomenclature scheme of previous studies (20,22,23) A, B, H, and N genotypes were found among the studied isolates.

Results
The phenotypic and biochemical properties of all 26 isolates indicated that they were all S. aureus strains. PCR amplification of the aroA gene and subsequent agarose gel electrophoresis of the amplified products showed an expected 1,153-bp amplicon for all S. aureus tested (Figure 1). No PCR product was amplified when sterile water was added instead of nucleic acids. Digestion of the PCR products with the TaqI restriction enzyme generated four different aroA gene patterns (Figure 2). In total, aroA type N was identified in 21 out of 26 (80.77%) isolates. aroA types A, B, and H were observed in 1 (3.85%), 2 (7.69%), and 2 (7.69%) isolates, respectively. The aroA types found for the strains of S. aureus coming from skin and urine samples are detailed in Table 1. As shown, all of the urine originating S. aureus isolates showed aroA type N. Different aroA types were identified among skin isolates, from which aroA type N was accounted for 5 (50%) isolates.

Discussion
Rapid identification of S. aureus DNA not only from bacterial cells but also from biological materials necessitates species-specific and ubiquitous nucleotide sequence as a target (24,25). In the current study, aroA gene was amplified from all tested S. aureus isolates.  Marcos et al (1999) described a rapid, sensitive, and specific nucleic acid-based procedure that permits the identification of S. aureus in cows and sheep with intramammary infections by PCR amplification of the aroA gene (20). Since then, PCR amplification of the aroA gene in combination with RFLP analysis has successfully been used for S. aureus identification and typing (22,23). According to the results of the present investigation, TaqI digestion of the PCRgenerated products from skin-related isolates revealed four different patterns A, B, H, and N, from which aroA type N was dominant, indicating its widespread existence. This result is in agreement with the results from Jordan, where fifty percent of the isolates collected from human specimens were designated as N (23), but is controversial when compared with earlier observation where aroA type N was not detected among the 59 S. aureus isolates from humans (20). In the present study, genotypes C and D were not detected among the tested isolates, but studies in Spain showed that 38.98% of the S. aureus isolates from human belonged to type A. Types B, C, and D were 13.5%, 3.3% and 44.1%, respectively (20). This is in agreement with that reported previously from the same region in Iran, where C and D aroA types were not detected among the bovine mastitis associated S. aureus isolates (22). These indicate that aroA genotypes were not distributed uniformly among various geographical areas, which may be related to different environmental conditions and the genetic background of the strain. More detailed studies including sequencing of the repeated region of the protein A gene (spa) to explore the area specific trait of S. aureus isolates will be required. As reported, the spa types among MRSA strains are area specific. spa type t002 is the most common type in Occupied Palestine (26), whereas spa type t041 is the common strain type in Southern Germany (27). Another well-known CA-MRSA is spa type t044, which is widespread in European countries (28)(29)(30). In a study from Austria, the most common spa types found among 382 MRSA isolates were t001, t190, t008, and t041 (31). Two spa types (t067 and t002) were dominant among MRSA strains in Spain (32).
As results, skin originated S. aureus isolates showed more genetic diversity than the urine ones. In a study from china, MSSA strains from skin/soft tissue infections in a children's hospital also showed diverse genetic backgrounds (33). Gadepalli et al (2009) also reported that different clones were responsible for all cases of SSTIs (12).
Considering the results from the current study and aroA RFLP patterns reported previously by Saei et al (2010) among the bovine mastitis-related S. aureus isolates, shows that the frequency of genotypes found for isolates of S. aureus from cow were in contrast to those found for isolates from human. One possible explanation for this is a host-and/or tissue-specific pathogenic trait of S. aureus genotypes may be due to genetic background. Apparently, the nature of the virulence genes encountered in an S. aureus strain is primarily an important determinant for host or even tissue specificity (11). Population genetic analyses have also provided strong evidence of host specialization among S. aureus clonal groups associated with human and ruminant infection (34). Although studies of the molecular epidemiology of S. aureus strongly suggest that a genetic subset of strains is particularly well adopted for causing infection in cattle (35)(36)(37), the distinct bovine clone groups are often interspread among the human clone groups in a manner suggesting that human-associated S. aureus clones were the evolutionary precursors to modern bovine specialist S. aureus clones (38,39).
As a result, aroA types A, B and H were only detected among skin associated isolates.
Existence of such a divergence shows that there is a possibility of skin contamination from various sources likely due to frequent person-to-person contact, contamination of surfaces, and sharing of personal hygiene items. This may also be as a consequence of the presence of certain virulence determinants and subsequence interaction between S. aureus and skin defense mechanisms. The first step in the establishment of S. aureus infection is adhesion to host tissues. S. aureus nasal and skin isolates produce Fibronectin Binding Protein-A (FnbPA) at a high frequency (40). It has been shown that IsdA (iron-responsive surface determinant A) allows S. aureus to colonize skin by blocking the action of several antibacterial molecules present in normal skin (41). Expanded studies are necessary to trace the origin of various genotypes of S. aureus that infect human skin. The significance of nasal carriage of S. aureus as risk factor for human skin infection has been reported previously (42,43). Tulloch (1954) reported that Staphylococci isolated from skin lesions in patients with chronic Staphylococcal skin infections were of the same phage-type as the Staphylococci isolated from anterior nares of the patients (44). MRSA nasal carriage, identified in 0.2% to 2.8% of the United States population, has been recognized as a risk for MRSA SSTI (45).
As indicated, only aroA type N was detected among the isolates originated from urinary tract infections. This type might be more adapted to the urinary tract and has special properties that play important role in its physiopathology. Baba-Moussa et al (2008) concluded that staphylococcal toxins and adhesion factors may be involved in the pathogenesis of S. aureus isolated from urinary tract infections (UTIs) (46). In vitro, S. aureus is able to attach to and aggregate onto uroepithelial cells through glycoproteins found in bladder mucin, like GP51, which is significantly increased in the presence of UTI (47). S. aureus harboring the Panton-Valentine Leukocidin (PVL) gene has been detected in urine, and this gene has been implicated in many different types of infections, including UTIs (48). Park et al (2008), believed that the icaA genes may enhance the adherence of S. aureus to host cells of the urinary tract, and may play a pathogenic role of UTI. Other speculation for this may be because of intermediate discriminatory power of aroA-gene based typing. In other study carried out by authors, this collection of S. aureus isolates were genotyped into six coa genotypes (C1-C6) using PCR-RFLP analysis.
In that study, 16 urine isolates with the same aroA type (N) differentiated to four coa RFLP profile (unpublished data). However, the combined use of coa and aroA PCR-RFLP techniques demonstrated that most (71.4%) isolates of genotype N clustered separately within coa type C1 (unpublished data). This result rather supports the finding of El-Huneidi et al (2006), who confirmed that N is a separate genotype (23). Whole genome sequencing and comparative genomic analysis may be useful to characterize the molecular genetic features that distinguish N type optimized for skin and urinary tract infections in human from those that infect bovine hosts or are only infrequently recovered from human sources. As suggested by other researchers (22,49), the correct epidemiological typing of S. aureus might require a combination of methods.

Conclusion
A very limited divergence between the isolates included in this study was described using aroA-gene based typing from two hospitals in the city of Urmia, Iran. However, the skin related isolates, were genetically divergence, may signifying cross-infection with S. aureus between humans and various sources such as environment. Here we also identified aroaA types which did not detected among S. aureus isolates recovered from UTIs. Most likely, the presence (combination) of virulence factors plays an important role in tissue specificity in S. aureus infections. This study also indicates that aroA genotypes vary not only from region to region, but also in individual host within a region. Further studies on these aspects from different regions of the country from time to time will help to monitor clonal dynamics of S. aureus in Iranian hospitals.